WO2022223548A1 - Composé et procédé pour la prophylaxie et le traitement de la leucémie - Google Patents

Composé et procédé pour la prophylaxie et le traitement de la leucémie Download PDF

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WO2022223548A1
WO2022223548A1 PCT/EP2022/060294 EP2022060294W WO2022223548A1 WO 2022223548 A1 WO2022223548 A1 WO 2022223548A1 EP 2022060294 W EP2022060294 W EP 2022060294W WO 2022223548 A1 WO2022223548 A1 WO 2022223548A1
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aml
leukemia
baalc
cells
inhibitor
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PCT/EP2022/060294
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Julia Skokowa
Benjamin Dannenmann
Maksim Klimiankou
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Eberhard Karls Universitaet Tuebingen Medizinische Fakultaet
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Priority to EP22716274.0A priority Critical patent/EP4326870A1/fr
Publication of WO2022223548A1 publication Critical patent/WO2022223548A1/fr
Priority to US18/489,480 priority patent/US20240122882A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention is directed to a compound for use in the prophylaxis and/or treatment of leukemia and/or the development of leukemia, a pharmaceutical com position for the prophylaxis and/or treatment of leukemia and/or the development of leuke mia, comprising said compound, a method for the prophylaxis and/or treatment of leuke mia and/or the development of leukemia in a living being, a single guide RNA (sgRNA) molecule which can be used in the method according to the invention.
  • sgRNA single guide RNA
  • the present invention relates to the field of molecular medicine, more particular to the field of small molecules and of genetic engineering applications, prefera bly to the targeted knockout of disease-associated genes.
  • Leukemia also spelled leukaemia, is a group of blood cancers that usu ally begin in the bone marrow and result in high numbers of abnormal blood cells. These blood cells are not fully developed and are called blasts or leukemia cells. Symptoms may include bleeding and bruising, fatigue, fever, and an increased risk of infections. These symptoms occur due to a lack of normal blood cells. Diagnosis is typically made by blood tests or bone marrow biopsy. [0004] The exact cause of leukemia is unknown. A combination of genetic fac tors and environmental (non-inherited) factors are believed to play a role. Risk factors in clude smoking, ionizing radiation, some chemicals (such as benzene), prior chemother apy, and Down syndrome. People with a family history of leukemia are also at higher risk.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myeloid leukemia
  • leukemia was present in 2.3 million people worldwide and caused 353,500 deaths. In 2012 it newly developed in 352,000 people. It is the most com mon type of cancer in children, with three-quarters of leukemia cases in children being the acute lymphoblastic type. However, over 90% of all leukemias are diagnosed in adults, with CLL and AML being most common in adults. It occurs more commonly in the devel oped world.
  • Severe congenital neutropenia is a pre-leukemia syndrome that, in the majority of patients, is caused by heterogeneous ELANE mutations encoding neutro phil elastase (NE).
  • the hematopoietic stem and progenitor cells (HSPCs) of CN patients fail to differentiate into neutrophilic granulocytes, without any severe maturation defects in other blood lineages.
  • the cumulative incidence of MDS or AML in CN patients is approxi mately 20% after 20 years.
  • Exposure of CN-HSPCs to high concentrations of granulocyte colony-stimulating factor (G-CSF) partially reverses granulocytic maturation defects. How ever, there is a correlation between susceptibility to G-CSF therapy and frequency of leu kemia.
  • Treatment of leukemia may involve some combination of chemotherapy, radiation therapy, targeted therapy, and bone marrow transplant, in addition to supportive care and palliative care as needed. Certain types of leukemia may be managed with watchful waiting. The success of treatment depends on the type of leukemia and the age of the person. Outcomes have improved in the developed world. Five-year survival rate is 57% in the United States.
  • This object is met by the provision of an inhibitor of 'mitogen-activated protein kinase-activated protein kinase 2' (MK2 inhibitor) for use in the prophylaxis and/or treatment of leukemia and/or the development of leukemia.
  • MK2 inhibitor 'mitogen-activated protein kinase-activated protein kinase 2'
  • mitogen-activated protein kinase-activated protein kinase 2 also designated as MK2, MAPKAPK2, MAPKAP-K2, MK-2, mitogen-ac tivated protein kinase-activated protein kinase 2, or MAPK activated protein kinase 2, re fers to an enzyme that in humans is encoded by the MAPKAPK2 gene.
  • This gene en codes a member of the Ser/Thr protein kinase family.
  • This kinase is regulated through di rect phosphorylation by p38 MAP kinase.
  • this kinase is known to be involved in many cellular processes including stress and inflammatory re sponses, nuclear export, gene expression regulation and cell proliferation.
  • MK2 inhibitor refers to a molecule which specifically inhibits the activity of MK2.
  • the MK2 inhibitor is a small molecule or an organic compound having a molecular size of ⁇ 900 daltons.
  • Exam ples of MK2 inhibitors are CMPD1, PF 3644022, PHA 767491 hydrochloride, MK2-IN-1 hydrochloride, MK2-IN-3 hydrate, CDD-450, MK2 inhibitor IV (MK 25; CAS no: 1314118- 94-9) etc.
  • the MK2 inhibitor can be a peptide or a small protein.
  • an existing leukemia can be effec tively treated by the MK2 inhibitor.
  • the inventors have also realized that the development of leukemia can be prevented by the use of an MK2 inhibitor.
  • This is of particular im portance because leukemia is often the result of a pre-existing condition such as pre-leu kemia bone marrow failure syndromes e.g., congenital neutropenia (CN), familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) or myelodysplas- tic syndrome (MDS), secondary therapy-related leukemias or others.
  • the invention also effectively remedies this situation and prevents the development of leukemia due to such pre-existing conditions.
  • the invention therefore, includes also the use of an MK2 inhibitor to prevent a relapsed leukemia, in particular relapsed AML.
  • the MK2 inhibitor can be used to prevent or reduce the uncontrolled proliferation of AML blasts, in e.g. case of recurrence or in the period between diagnosis and bone marrow transplantation.
  • the MK2 inhibitor can be used as the only active agent, i.e. in form of a monotherapeutics, without any additional further active agent. It is understood that the anti-leukemia activity according to the inven tion is exerted by the MK2 inhibitor.
  • a synergistic activ ity on an other compound which is used against leukemia e.g. SAHA (vorinostat) is ex plicitly excluded and not subject of the present invention.
  • MK2 inhibitors are described in relation with several tumors, such as glioblastoma (Gurgis et al. , 2015, Cytotoxic activity of the MK2 inhibitor CMPD1 in glioblastoma cells is independent of MK2, Cell Death Discovery 1, 15028, p. 1- 11), or gastric cancer (Li et al., 2018, CMPD1 inhibited human gastric cancer cell prolifera tion by inducing apoptosis and G2/M cell cycle arrest, Biol. Res. 51:11, p. 1-9).
  • CMPD1 does not affect the regula tion of several canonical MK2 targets at doses that are potently synergistic in AML cell lines, including the phosphorylation of HSP27 or AATF, and rather teach away from the invention.
  • said leukemia is acute myeloid leukemia (AML).
  • This measure has the advantage that one of the most clinically relevant forms of leukemia is effectively addressed, which the art fails so far in doing so.
  • This em bodiment includes all different kinds of AML, such as de novo AML, RUNX1-mutated AML, secondary therapy-related AML etc.
  • said AML is de novo AML.
  • the inventors have surprisingly realized in an experimental set-up that for variants of AML which occur independently and without prior bone marrow disease or cancer, the administration of an MK2 inhibitor is of particular effectiveness.
  • said AML is AML with high ex pression of BAALC (BAALC hi9h ).
  • BAALC refers to a gene that codes for the 'brain and acute leukemia cytoplasmic' protein (human variant: NCBI gene no. 79870).
  • the function of BAALC is not fully understood yet but high BAALC expression level is known to be associated with more aggressive AML and has been described to have ad- verse impact on the outcome of AML; see Baldus et al. (2003), BAALC expression pre dicts clinical outcome of de novo acute myeloid leukemia patients with normal cytogenet ics: a Cancer and Leukemia Group B Study, Blood 102(5), p. 1613-1618; Baldus et al.
  • BAALC expression a suitable marker for prognos tic risk stratification and detection of residual disease in cytogenetically normal acute mye loid leukemia, Blood Cancer J. 4(1):e173. This measure has the advantage that a particu larly aggressive form of AML can be effectively treated and/or prevented by the invention.
  • said development of leukemia results from a pre-existing condition that is selected from the group consisting of: congeni tal neutropenia (CN), familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) or myelodysplastic syndrome (MDS).
  • CN congeni tal neutropenia
  • FPD/AML familial platelet disorder with predisposition to acute myelogenous leukemia
  • MDS myelodysplastic syndrome
  • the indicated conditions are well-known for pre-existing diseases which are high risk factors for a subsequent development of AML.
  • the inven tion can be effectively used in an early stage prior to or in the development of AML to eliminate or at least significantly reduce the risk of a progression of the pre-existing condi tions.
  • the invention can even be used for the prophylaxis and/or treatment of the indi cated pre-conditions, thereby effectively preventing the manifestation of leukemia or AML, respectively.
  • the MK2 inhibitor is an MK2a in hibitor.
  • This measure has the advantage that such an isoform of MK2 is specifi cally addressed by the invention which plays a central role in the p38MAPK/MK2- dependent signaling pathway and, therefore, in the development of leukemia.
  • the MK2 inhibitor is CMPD1.
  • CMPD1 (CAS. no.: 41179-33-3; MW: 349.4, C22H20FNO2) is a selective and non-ATP-competitive p38 MAPK-mediated MK2 phosphorylation inhibitor with appar ent Ki (Ki aPP ) of 330 nM.
  • CMPD1 induces a gene expression profile which is similar to the one that can be observed when knocking out the BAALC gene, thereby reversing the driving force of the development of AML.
  • CMPD1 is, therefore, according to the findings of the inventors, one of the most effective MK2 inhibitors in the prophylaxis and treatment of leukemia, in particular of AML.
  • Another subject-matter of the invention is a pharmaceutical composition for the prophylaxis and/or treatment of leukemia and/or the development of leukemia, comprising the MK2 inhibitor according to the invention and a pharmaceutically accepta ble carrier.
  • a "pharmaceutical composition” is a composition suitable for an admin istration to an animal and/or human being in a medical setting.
  • a pharmaceuti cal composition is sterile and produced according to GMP guidelines.
  • the MK2 inhibitor can be used as the only active agent of the pharmaceutical composition, i.e. in form of a monotherapeutics, without any additional further active agent.
  • the pharmaceutical composition comprises the MK2 inhibitors either in the free form or in the form of a pharmaceutically acceptable salt.
  • Pharmaceutically acceptable carriers are well known in the art and in clude, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable or ganic esters.
  • aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable or ganic esters.
  • the aqueous solution is pyrogen- free, or substantially pyrogen-free.
  • the excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (in cluding sprinkle capsule and gelatin capsule), granule, powder, syrup, suppository, injec tion or the like.
  • the composition can also be present in a transdermal delivery system, e.g., a skin patch.
  • the composition can also be present in a solution suitable for topical administration, such as an eye drop.
  • the pharmaceutical composition can be present in form of an aerosol administrable by inhalation.
  • the pharmaceutical composition as well as the methods to be explained below of the present invention may be utilized to treat a living being in need thereof.
  • the living being is a mammal such as a human, or a non-human mammal.
  • the MK2 inhibitor is present per dosage unit at a concentration which, upon administration into a living being, results in a concentration in said living being which inhibits the leukemogen- esis and/or eliminates leukemia cells in said living being.
  • This measure has the advantage that the MK2 inhibitor is provided in said pharmaceutical composition in an adequate amount, thereby reducing potential side effects to a minimum.
  • the invention further relates to a method for the prophylaxis and/or treat ment of leukemia and/or the development of leukemia in a living being, preferably a hu man being, comprising a step of inhibiting the leukemogenesis in said living being, wherein said inhibition of the leukemogenesis and/or elimination of leukemia cells is real ized via the administration of an MK2 inhibitor to said living being.
  • the invention also relates to a method for the prophylaxis and/or treat ment of leukemia and/or the development of leukemia in a living being, comprising a step of inhibiting the leukemogenesis in said living being, wherein said inhibition of the leuke- mogenesis is realized via the inhibition of BAALC in said living being, preferably knocking out of the gene encoding BAALC (BAALC KO) in said living being.
  • inhibittion of BAALC refers to any phenome non which specifically and selectively reduces or abolishes the activity and/or functionality of BAALC in said living being, e.g. human being.
  • This inhibition can be achieved, e.g. via the administration into the living being of a specific BAALC inhibitor, an siRNA inhibiting the translation of the coding molecule of BAALC, or, preferably, via knocking out the gene encoding BAALC (BAALC KO) etc.
  • the BAALC KO is realized via the use of proteases, preferably via CRISPR/Cas9 gene-editing.
  • This method has the advantage that the BAALC gene is knocked-out in a targeted manner, thereby limiting or even avoiding off-target effects.
  • the invention further relates to a 'single guide RNA' sgRNA molecule targeting the gene encoding BAALC in a living being, preferably a human being, for use in the prophylaxis and/or treatment of leukemia and/or the development of leukemia.
  • a tool which can be used in the method ac cording to the invention for knocking out of the gene encoding BAALC via CRISPR/Cas9 gene-editing.
  • a "single guide RNA” (sgRNA) or guide RNA is a component of the CRISPR complex which serves to guide the CRISPR endonuclease to its target.
  • the sgRNA is a non-coding short RNA sequence which binds to the comple mentary target DNA sequence of the BAALC gene.
  • the sgRNA first binds to the CRISPR endonuclease enzyme and the sgRNA sequence guides the complex via pairing to the lo cation on the DNA specifically encoding the BAALC gene. There the modified CRISPR endonuclease performs its nickase activity by cutting the target single DNA strand result ing in a knock-out of the BAALC gene.
  • Another subject-matter of the invention is a nucleic acid molecule, pref erably the sgRNA molecule according to the invention, comprising any of the nucleotide sequences of SEQ ID NOS:1, 2, 39-78.
  • nucleic acid molecule pre cisely cuts the BAALC encoding sequence on the DNA, thereby ensuring a knocking out of the BAALC gene resulting in the targeted inhibition of the leukemogenesis.
  • Fig. 1 Allele frequencies of missense RUNX1 mutations in CN/AML with trisomy 21.
  • A Analysis of the allele frequency (AF) of wild type (WT) and corresponding mutant RUNX1 in CN patients prior- and after onset of AML (CN/AML). Each bar represents percentage of WT (green) and mutant (red) RUNX1 AF quanti fied by dPCR.
  • genotyping of BM or PB samples revealed an absence of mutant RUNX1 allele; whereas, in overt AML, 59-68% of leukemia cells acquired RUNX1 mutations, indicating the gain of a second mutant RUNX1 allele. Data are represented as mean ⁇ SD.
  • FIG. 1 A stepwise in vitro model of leukemogenesis in congenital neutropenia through the generation of patient-specific iPSCs and the insertion of additional mutations using CRISPR/Cas9 gene-editing.
  • Top panel represents the time scale of leukemic transformation in CN/AML patients, the bottom panel represents the genera tion of single iPSC clones at specific time points during disease progression.
  • C dPCR analysis of the allele frequency of WT and mutant (p.R139G)
  • Fig. 2 Representative images of digital PCR data plots for quantification of RUNX1 allele ratio in CN and CN/AML samples, related to Figure 1.
  • the x-axis shows the signal from FAM reporter dye, and the y-axis shows the - the VIC reporter dye.
  • Blue dots represent FAM-labeled mutant RUNX1 PCR product
  • red dots represent VIC-labeled WT RUNX1 PCR product
  • green dots repre sent droplets containing both mutant and WT DNA
  • yellow dots are drop lets with no DNA incorporated. Ratio of mutant and WT RUNX1 allele was cal culated based on number of FAM and VIC positive signals on the chips.
  • Pat.#1 DNA isolated from BM (CN-phase) and DNA extracted from single-cell derived CFU clones (CN/AML phase) were amplified using TaqMan assay probe set against WT RUNX1 and RUNX1 p.R139G allele.
  • Fig. 3 Genetic analysis of iPSCs clones derived from CN/AML patients, related to
  • FIG. 1 and 6 Representative Sanger Sequencing diagrams of DNA iso lated from indicated iPSC clones confirming ELANE p.C151R, CSF3R p.Q741X and RUNX1 p.R139G heterozygous mutations for CN/AML patient 1, and ELANE p.G214R and CSF3R p.Q743X heterozygous mutations for CN/AML patient 2.
  • B Representative images of array CGH of indicated iPSC clones. Whole genome view is shown.
  • C-E Assessment of CRISPR/Cas9 gene-edited iPSC clones.
  • DNA isolated from single-cell derived iPSC clones was amplified by PCR and analyzed using TIDE (Tracking of Indels by DE- composition). Graphs show the estimated composition of the inserted/deleted (in/del) nucleotide bases in iPSC lines calculated by TIDE. Additionally, ge netic changes introduced by CRISPR/Cas9 have been annotated at protein level in order to predict the consequence of in/del introduced by genome edit ing in the amino acid sequences of modified genes.
  • Fig. 4 Characterization of HD, CN and CN/AML iPSC clones, related to Figure 6.
  • B qRT-PCR analysis of mRNA expression of pluripotency-specific genes us ing mRNA isolated from indicated iPSC clones of CN/AML patient 1 (A) and patient 2 (B). Gene expression levels were normalized to GAPDH and shown relative to HD CD34+ cells. PRE indicates exogenous mRNA expression of the lentiviral vector. Data are represented as mean ⁇ SD; two independent ex periments.
  • C Percentage of pluripotency-specific surface markers (Tra1-60 and SSEA4) in indicated iPSC clones of CN/AML patient 1 and 2.
  • D, E Rep resentative images of the Alkaline Phosphatase activity (purple) analysis in iPSC colonies of CN/AML patient 1 (D) and patient 2 (E).
  • Fig. 5 Analysis of hematopoietic differentiation of CN and CN/AML iPSC-derived
  • HSPCs by flow cytometry related to Figure 6.
  • A Schematic overview of the EB-based hematopoietic/neutrophilic differentiation of iPSCs.
  • B, C Percent age of iPSC-derived immature hematopoietic cells (CD34 + KDR + ,
  • iPSCs were cultured under EB differentiation conditions for 14 days. Data are represented as mean ⁇ SD; two independent experiments; including group comparisons. Significant difference between groups to CN is indicated, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Fig. 6 Hematopoietic differentiation of CN/AML patients derived iPSCs.
  • A, B Per centage of CD34 + cells derived from CN/AML1 (A) and CN/AML2 (B) iPSCs on day 14 of culture. Data are represented as mean ⁇ SD; two independent experiments. Group comparisons were done between CN and all other iPSC clones. Significant difference between groups to CN is indicated, *P ⁇ 0.05,
  • C Proliferation rate of CD34 + cells derived from CN/AML1 (C) and CN/AML2 (D) iPSCs on day 14 of culture.
  • HSPCs were expanded on SL/SL feeder for 7 days. Data were normalized to healthy donor (HD) control and are represented as mean ⁇ SD; two independent experiments. Significant difference between groups to CN is indicated, *P ⁇ 0.05, **P ⁇ 0.01.
  • E, F Percentage of iPSC-derived granulocytes (CD15 + CD16 + CD45 + and CD15 + CD11 b + CD45 + ) in CN/AML1 (E) and CN/AML2 (F).
  • iPSCs were cultured under EB differentiation conditions for 32 days. Data are represented as mean ⁇ SD from two independent experiments. Group comparisons were done be tween CN and all other iPSC clones. Significant difference between groups to CN is indicated, *P ⁇ 0.05, **P ⁇ 0.01.
  • G, H Representative images of iPSC- derived hematopoietic cells at day 32 of differentiation stained with Wright Giemsa are shown for CN/AML1 (G) and CN/AML2 (H). 40x magnification is shown.
  • I, J CFU assay of iPSC-derived CD34 + cells isolated from EBs cul ture at day 14.
  • Fig. 7 Up-regulation of AML-associated genes in primary CN/AML blasts and
  • CN/AML iPSC-derived CD34 + cells CN/AML iPSC-derived CD34 + cells.
  • A mRNA expression of indicated genes in BMMNCs from three CN and five CN/AML patients, measured by qPT-PCR. Gene expression levels were normalized to b-actin and shown relative to CN. Data are represented as mean ⁇ SD from two independent experiments. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 compared to CN.
  • B, C mRNA expression of indicated genes in CD34 + cells derived from CN1 and CN/AML1 (B) as well as CN2 and CN/AML2 (C) iPSCs, measured by qPT-PCR.
  • (E) mRNA expres sion of BAALC in CD34 + cells from healthy individuals (n 2) transduced with control GFP, WT RUNX1 or mutant RUNX1 (p.R139G and p.R174L) lentiviral constructs, measured by qPT-PCR. Gene expression levels were normalized to b-actin. Data are represented as mean ⁇ SD from two independent experi ments. *P ⁇ 0.05.
  • Fig. 8 BAALC upregulation is essential for leukemogenic transformation in CN/AML.
  • C Percentage of iPSCs derived granulocytes (CD15 + CD16 + CD45 + and CD15 + CD11b + CD45 + ) on day 32 of culture. Data are represented as mean ⁇ SD from two independent experiments. *P ⁇ 0.05.
  • D Representative images of iPSC-derived hematopoietic cells at day 32 of differentiation stained with Wright Giemsa at 40x magnification are shown.
  • E CFU assay of iPSCs-de- rived CD34 + cells isolated at day 14 of culture. Total CFU counts are shown. Data are represented as mean ⁇ SD from two independent experiments in trip licate. Group comparisons were done between CN/AML1 and all other iPSC clones.
  • CD15+CD11b+CD45+ on day 32 of culture.
  • Data are represented as mean ⁇ SD from two independent experiments. *P ⁇ 0.05, **P ⁇ 0.01.
  • K Proliferation rate of primary CN/AML mock and CN/AML BAALC KO blasts from three CN/AML patients. Gene-edited primary CN/AML blasts were expanded on SL/SL feeder for 14 days. Data were normalized to mock cells and are repre sented as mean ⁇ SD, *P ⁇ 0.05.
  • Fig. 9 Assessment of CRISPR/Cas9 gene-edited knockout iPSC lines and further analysis of hematopoietic differentiation in CN/AML iPSC knockout lines, re lated to Figure 8.
  • A-F, l-J DNA isolated from single-cell derived iPSC clones was amplified by PCR and analyzed using TIDE (Tracking of Indels by De composition). Graphs show the estimated composition of inserted/deleted (in/del) nucleotide bases in iPSC lines calculated by TIDE.
  • ge netic changes introduced by CRISPR/Cas9 have been annotated at protein level in order to predict the consequence of in/dels introduced by genome edit ing in the amino acid sequences of modified genes.
  • G, H Percentage of iPSC-derived immature hematopoietic cells (CD34 + KDR + , CD34 + CD43 + ,
  • iPSCs were cultured under EB differentiation condi tions for 14 days. Data are represented as mean ⁇ SD; two independent ex periments; including group comparisons. Significant difference between groups to CN is indicated, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • K Alignment of Sanger sequencing traces from parent control iPSC line (upper panel) and base edited HD BAALC KO (lower panel) confirmed the introduction of BAALC stop codon mutation in a single-cell derived iPSC clone. Positions of based edited nucleotides are depicted with black arrows. PAM sequence of guide RNA is marked above the alignment with a red line.
  • Fig. 10 Gene expression signature of CD34 + cells derived from CN and CN/AML iP SCs.
  • A, B Volcano plot showing differentially expressed genes (DEGs, log2FC > 1 or ⁇ -1, adjusted P-value ⁇ 0.05) in iPSC-derived CD34 + cells from (A) CN/AML1 vs CN1 and (B) CN/AML2 vs CN2 group comparisons.
  • the x- axis shows the log2 fold change, and the y-axis shows the -Iog10 adjusted P- value. Colors represent the significance of the genes in terms of P-value and log fold change.
  • Gene names are indicated for the top 10 upregulated and top 10 downregulated genes.
  • Fig. 11 Gene expression and histone H3 trimethyl Lys27 (H3K27me3) analysis of iPSC-derived HSPCs, related to Figure 10 and 12.
  • (A) Supervised average linkage clustering of 702 differentially expressed genes (log FC > 1 or ⁇ -1, adj. P-value ⁇ 0.05) between iPSC-derived HSPCs from CN/AML1 and CN1 RNA-seq samples were obtained from 3 independent EB-based hematopoietic differentiation experiments performed with CN1, CN/AML1.1, or CN/AML1.2 iPSC clones.
  • Expression heatmap was created using variance stabilizing transformed read counts as an input.
  • Fig. 12 BAALC-dependent gene expression profile of CN/AML iPSC-derived CD34 + cells.
  • A, B Volcano plot showing DEGs in iPSC derived CD34 + cells from (A) CN/AML1 vs CN/AML1 BAALC KO and (B) CN/AML2 vs CN/AML2 BAALC KO group comparisons.
  • the x-axis shows the log2 fold change, and the y- axis shows the -Iog10 adjusted P-value. Colors represent the significance of the genes in terms of P-value and log2 fold change.
  • Gene names are indicated for the top 10 upregulated and top 10 downregulated genes.
  • Data are displayed as -Iog10 of adjusted P-value.
  • I, J Venn diagram showing overlap of transcription fac tors (I) and kinases (J) between CN/AML vs CN/AML BAALC KO (pink) and CN/AML vs CN (blue) groups of both CN/AML patients.
  • Fig. 13 MK2a- and p38a- inhibitor treatment of iPSC-derived CD34 + cells, primary
  • CN/AML blasts and de novo AML blasts related to Figure 14.
  • A Connectivity MAP analysis of RNA-seq data of CN/AML and CN/AML BAALC KO iPSC-de- rived CD45 + CD34 + cells. pc_selection represents the percent of total perturba- gens, querying the column sample against selected rows that exceed the given thresholds.
  • B Flow cytometry analysis of intracellular MK2a and p38a levels in CN, CN/AML, CMPD1-treated CN/AML (1 mM of CMPD1 for 1 day), and CN/AML BAALC KO iPSC-derived CD34 + CD45 + cells.
  • Data are repre sented as mean ⁇ SD; two independent experiments of both patients.
  • C Pro liferation rate of Kasumi-1 WT cells treated with either AZD-6244 (1, 2 and 5 pM), CMPD1 (1, 2 and 5 pM), or DMSO for 7 days. Data are represented as mean ⁇ SD; two independent experiments. *P ⁇ 0.05, ****P ⁇ 0.0001 com pared to DMSO control.
  • D Proliferation rate of Kasumi-1 WT cells treated with U0126 (1, 5 and 10 pM) or DMSO for 7 days. Data are represented as mean ⁇ SD; two independent experiments.
  • E Proliferation rate of HD, CN, and CN/AML iPSC-derived CD34 + CD45 + cells treated with either 1pM AZD- 6244 or DMSO for 7 days. Data are represented as mean ⁇ SD; two independ ent experiments. *P ⁇ 0.05, **P ⁇ 0.01 compared to DMSO control.
  • F Prolif eration rate of primary CN/AML blasts treated with either 1 pM AZD-6244 or DMSO for 7 days. Data are represented as mean ⁇ SD; two independent ex periments. **P ⁇ 0.01 compared to DMSO control.
  • Fig. 14 Inhibition of proliferation of iPSC-derived and primary CN/AML or de novo
  • CM PD 1 -treated CN/AML (1 pM CMPD1; 24 hours), and CN/AML BAALC KO iPSC-derived CD34+ cells Data are represented as mean ⁇ SD from two in dependent experiments of two CN/AML patients. * P ⁇ 0.05, **P ⁇ 0.01 , ***P ⁇ 0.001 compared to CN/AML.
  • B Proliferation rate of iPSC-derived CD34+ cells from HD, CN and CN/AML clones treated with either 1 pM CMPD1 or DMSO for 7 days. Data are represented as mean ⁇ SD from two independent experiments. **P ⁇ 0.01 compared to DMSO control.
  • FIG. 1 Schematic diagram of the role of BAALC upregulation in CN-related leukemia development.
  • CN patients with inherited CN-associated mutations e.g., ELANE
  • genetically ‘unfit’ HSPCs acquire CSF3R mutations.
  • the acquisition of CSF3R mutations along with prolonged exposures to high doses of G- CSF leads to the clonal selection of pre-leukemia HSPCs, which exhibit elevated proliferation and reduced differentiation upon G-CSF treatment.
  • the coacquisition of further RUNX1 haploinsufficiency or missense RUNX1 mutations along with trisomy 21 leads to elevated BAALC expression levels and leuke- mogenic transformation.
  • Fig. 15 In vivo analysis of the effects of MK2 inhibition on the proliferation and survival of primary CN/AML patients blasts xeno-transplanted into zebrafish embryos.
  • Pre-leukemia bone marrow failure syndromes are characterized by ab normal differentiation and functions of hematopoietic stem and progenitor cells (HSPCs). They frequently culminate in the development of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML).
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • HSPCs Severe congenital neutropenia (CN) is a pre-leukemia syn drome that, in the majority of patients, is caused by heterogeneous ELANE mutations en coding neutrophil elastase (NE).
  • CN Severe congenital neutropenia
  • NE heterogeneous ELANE mutations en coding neutrophil elastase
  • the cu mulative incidence of MDS or AML in CN patients is approximately 20% after 20 years. Exposure of CN-HSPCs to high concentrations of granulocyte colony-stimulating factor (G-CSF) partially reverses granulocytic maturation defects. However, there is a correlation between susceptibility to G-CSF therapy and frequency of leukemia. The inventors re cently reported a high frequency of acquired cooperative mutations in CSF3R (encoding the G-CSF receptor) and RUNX1 (runt-related transcription factor 1) in 55% of CN/AML patients (31 patients were studied). Recently, the inventors described a clinical case of two cyclic neutropenia (CyN) patients with acquired CSF3R mutations.
  • CyN cyclic neutropenia
  • CSF3R mutations are stop-codon mutations localized in the intracellular domain of the G-CSF receptor, which is responsible for the inhibition of STAT5 mediated proliferative signals by SOCS3, and the activation of STAT3-dependent differentiation. Missense and nonsense RUNX1 mutations in the DNA-binding Runt ho mology domain (RHD), or truncating mutations in the transactivation domain (TAD) have been detected in CN/AML patients.
  • RHD Runt ho mology domain
  • TAD transactivation domain
  • RUNX1 maps to chromosome 21, and leukemic blasts from some CN/AML patients harbor trisomy 21. Thus, the ratio of mutant to wild type RUNX1 alleles may con tribute to leukemogenesis.
  • iPSCs patient-specific induced pluripotent stem cells
  • CRISPR/Cas9 gene-editing it is possible to introduce distinct mutations in iPSCs and to study stepwise, stage-specific leukemia progression. This approach allows the development of leukemia models to compare different types of mutations (e.g., missense versus nonsense) in en dogenously expressed proteins (e.g., RUNX1).
  • the inventors established an in vitro model of leuke mia evolution in CN using patients-derived iPSCs and CRISPR/Cas9-mediated introduc tion of gene mutations. Using this model, the inventors described the upregulation of BAALC (brain and acute leukemia, cytoplasmic) as a key leukemogenic event.
  • BAALC brain and acute leukemia, cytoplasmic
  • the inven tors identified a small molecule inhibitor of MK2 phosphorylation that targets CN/AML blasts without affecting healthy HSPCs. This strategy could be applied to treat CN/AML or de novo AML patients with RUNX1 mutations and/or elevated BAALC expression.
  • CN and CN/AML patient samples for reprogramming were received from Severe Chronic Neutropenia International Registry (SCNIR), Hannover, Germany. In formed written consent was obtained from all participants of this study. The experiments involving human inducible pluripotent stem cells (iPSCs) were performed with the ap proval obtained from the Ethical Review Board of the Medical Faculty, University of Tu bingen.
  • SCNIR Severe Chronic Neutropenia International Registry
  • Peripheral blood mononuclear cells or bone marrow mononu clear cells (BMMNCs) from 2 male CN/AML patients at different stages of leukemic trans formation and male healthy donors were reprogrammed using Oct4, Sox2 and KLF4 (OSK) lentivirus (pRRL.PPT.SF.hOct34.hKlf4.hSox2.i2dTomato.pre.FRT, kindly provided by A. Schambach, MHH Germany). Additional mutations in CSF3R or RUNX1 were intro Jerusalem using CRISPR/Cas9 gene-editing, if not already obtained by reprogramming.
  • iP SCs were cultured on SNL-feeder cells (Public Health England) for general maintenance or feeder-free on Geltrex with StemFlex medium (Thermo Fisher Scientific) for further gene-editing experiments.
  • PBMNCs were cultured for 6 days in CD34 + cells expansion me dium (Stemlinell medium, Sigma-Aldrich) supplemented with 10 % FCS, 1 % Pen/Strep, 1 % Glutamine and cytokines: IL-3 (20 ng/ml), II-6 (20 ng/ml), TPO (20 ng/ml), SCF (50 ng/ml) and FLT3L (50 ng/ml). All cytokines were purchased from R&D Systems. After 1 week, cells were transferred to Retronectin (Clontech)-coated 12-well plates together with OSK lentiviral supernatant at a multiplicity of infection (MOI) of 2.
  • MOI multiplicity of infection
  • iPSC-medium and CD34 + cell expansion medium supplemented with 2 mM valproic acid and 50 pg/ml Vitamin C.
  • Medium was gradually changed to iPSCs medium only.
  • First iPSCs colonies appeared ap proximately three weeks after initiation of reprogramming.
  • Genomic DNA of iPSCs was isolated using Nucleo-Spin Tissue Kit (Ma- chery-Nagel) and DNA regions for sequencing were amplified using the following primers: RUNX1-F 5'-ACATCCCT GAT GT CTGCATTT GTCC-3' [SEQ ID NO: 33], RUNX1-R 5'- T GT GGGTTT GTT GCCA TGAAACGTG-3' [SEQ ID NO: 34], ELANE-F 5'- CGCCCTGAGCCTTGGTGACG-3' [SEQ ID NO: 35], ELANE-R 5'- AGCCACGGTGCCTGTTGCTG-3' [SEQ ID NO: 36], CSF3R-F 5'- AT GGCAT GT GTCAGGCAT GT -3' [SEQ ID NO: 37], CSF3R- R 5'- AGTCACAGCGGAGATAGTGC-3' [SEQ ID NO: 38] Sanger Sequencing was performed by GATC Biotech. iPSCs culture
  • iPSCs were maintained on mitomycin-C treated SNL-feeder cells (Public Health England) in iPSC-medium consisting of DMEM F12 (Sigma-Aldrich) supplemented with 20 % Knockout Serum Replacement (Invitrogen), 30 ng/ml bFGF (Peprotech), 1 % non-essential amino acids solution (Invitrogen), 100 mM 2-Mercapto-Ethanol and 2 mM L- Glutamine. iPSC-medium was replaced every day. For CRISPR/Cas9 gene-editing experi ments and expansion of single cell derived clones, iPSC lines were cultured on Geltrex with StemFlex medium (Life Technologies).
  • sgRNAs capable of knocking out of the BAALC gene by targeting the first coding exon are listed in Table 3.
  • Corresponding sgRNAs (Table 2) were cloned into all-in one pSpCas9(BB)-2A-GFP (PX458) plasmid, a gift from Feng Zhang (Addgene plasmid # 48138).
  • CN- and CN/AML iPSC lines were nucleofected with 1-5 pg PX458- sgRNA construct using P3 Primary Cell 4D-NucleofectorTM X Kit L (Lonza) and 4D Nu- cleofector (Lonza) or reverse transfected with 7ranslT®-LT1 Transfection Reagent (Mirus).
  • BAALC KO in HD iPSCs was introduced by transfection with cytosine base editor plasmid BAALC_p.19.NLS.BE3.2xNLS.GFP.PX458 which was generated by sticky end cloning of BE3 into PX458 backbone followed by cloning guide RNA sequence BAALC p.19 (Table 2).
  • BE3 insert was isolated from pCMV-BE3 plasmid which was a gift from the David Liu lab (Addgene plasmid # 73021). GFP + cells were sorted 48 hours post-transfec- tion and cultured on the Geltrex. Single-cell clones were analyzed by Sanger sequencing at the gene-edited target regions.
  • BAALC KO in primary CN/AML cells was achieved by nucleofection of Cas9-sgRNA-BAALC-p.20 ribonucleoprotein (RNP) complex using P3 Primary Cell 4D-NucleofectorTM X Kit L (Lonza) and 4D Nucleofector (Lonza). The RNP nucleofection protocol has been recently published by our group. Off-target sites were predicted using http://crispor.org. The top 3 sites with the highest off-target scores and/or exon localization were selected for Sanger sequencing (Table 4).
  • iPSCs were dissociated from SNL-feeders or Geltrex (Thermo Fisher Scientific) coated plates using PBS/EDTA (0.02%) for 5 min.
  • EB generation was done via centrifugation of 20.000 cells per EB in 96-well plates using APEL serum-free differentia tion medium (Stemcell Technologies) supplemented with bFGF (20 ng/mI) and ROCK In hibitor Y-27632 dihydrochloride (Tocris).
  • APEL serum-free differentia tion medium Stem Technologies
  • bFGF 20 ng/mI
  • ROCK In hibitor Y-27632 dihydrochloride Tocris.
  • BMP4 40 ng/ml
  • EBs were plated on Matrigel-coated 6-well plates (10 EBs/well) in APEL medium supplemented with VEGF (40 ng/ml) (R&D Systems), SCF (50 ng/ml) (Peprotech) and IL-3 (50 ng/ml) (Peprotech).
  • VEGF 40 ng/ml
  • SCF 50 ng/ml
  • IL-3 50 ng/ml
  • G-CSF 50 ng/ml
  • 1-3x10 5 iPSC-derived CD45 + CD34 + cells, primary CN/AML blasts, or de novo AML blasts were cultured on SL/SL feeder cells producing FLT3L (kindly provided by C. Eaves, Vancouver, Canada) in HLTM/Myelocult H5100 medium (Stemcell Technolo gies) supplemented with 10 6 M hydrocortisone, IL-3 (20 ng/ml), II-6 (20 ng/ml), TPO (20 ng/ml), SCF (50 ng/ml) and FLT3L (50 ng/ml) for 7 days with medium change every 3 - 4 days.
  • CD34 + cells from healthy donors (2x10 5 /well) were transduced with lenti- viral supernatant at a MOI of 5.
  • a second transduction was performed the next day. Seventy-two hours post-trans-duction, GFP-positive cells were sorted and analyzed for RUNX1 and BAALC mRNA ex pression by qRT-PCR. Vector and primer information is available upon request.
  • the following primary antibody were used: anti-RUNX1/AML1 (Cell Signaling Technology, #4334, 1:500), anti- STAT5a (Cell Signaling Technology, #4807, 1:500) anti-G-CSFR (Santa Cruz Biotechnol ogy, #sc-74026, 1:500), and b-Actin (Cell Signaling Technology, #4970, 1:1000) and anti- BAALC (Santa Cruz Biotechnology, #sc-515606, 1:500).
  • membranes were washed and incubated with secondary HRP-coupled antibody (Cell Signaling Technology, #7076 or #7074, 1:2000) for 1 hour at room temperature.
  • Pierce ECL solution Thermo Fisher Scientific
  • Amersham Hyperfilm GE Healthcare
  • iPSC colonies on SNL-feeders at day 10 of culture were washed with PBS, fixed in 4 % PFA /10 % sucrose in water and stained with NBT/BCIP staining dye (Sigma-Aldrich) for 20 min at RT.
  • Array-CGH was performed using the Agilent Human Genome Microarray Kits 2 x 400K (Agilent Technologies). Labelling and hybridization of genomic DNA was performed according to the protocol provided by Agilent. Microarray slides were scanned using an Agilent microarray scanner G2505B at a resolution of 2 pm. For image analysis, default CGH settings of Feature Extraction Software (Agilent Technologies) were applied. Output files from Feature Extraction were subsequently imported into Agilent’s CGH data analysis software, Genomic-Workbench. The Aberration Algorithm ADM2 was applied and Aberration Filters were set to: threshold 7.0, at least 4 probes with mean log2 ratio of +/- 0.3 leading to a resolution of approximately 20 kb.
  • a mul ticolor FACS antibody panel for ‘early-stage’ hematopoietic differentiation using the follow ing antibody was applied: CD33-BV421 (BioLegend, BL), CD34-PeCy7 (BD Biosciences, BD), KDR-AF647 (BL), CD43-PE (BD), CD41a-FITC (BD), CD235a-FITC (BD), CD45- BV510 (BL), 7-AAD (BD).
  • a multicolor FACS anti body panel for ‘late-stage’ hematopoietic differentiation using the following antibody was applied: CD15-PE (BD), CD16-FITC (BD), CD14-APC-H7 (BD), CD45-BV510 (BL), CD33, BV-421 (BL), 7-AAD (BD).
  • CD15-PE CD15-PE
  • CD16-FITC CD14-APC-H7
  • BD CD45-BV510
  • BL CD33
  • BV-421 BL
  • 7-AAD 7-AAD
  • iPSCs characterization the stem cell surface markers TRA1-60-PE (eBioscience) and SSEA4-FITC (BD) were analyzed.
  • TRA1-85-APC R&D Systems
  • Anti-mouse IgGk beads BD were used for compensation. Samples were analyzed using FACSCanto II (BD) and FlowJo V10 (BD).
  • RNA-seq data in fastq files was assessed using FastQC (vO.11.4).
  • Reads were aligned using STAR (v2.4.2a) allowing gapped alignments to ac count for splicing against a custom-built genome composed of the Ensembl Homo Sapi ens genome v90. Alignment quality was analyzed using samtools (v1.1) and visually in spected in the Integrative Genome Viewer (v2.3.67). Read counts per gene were ex tracted with HTSeq count.
  • CLUE platform https://clue.io/cmap was used for CMAP analysis to evaluate the gene expression profiles of CN/AML and CN/AML BAALC KO RNA-seq data sets for connectivity to known perturbagens.
  • iPSC-derived CD45 + CD34 + cells were fixed for 15 min with 4% PFA and permeabilized for 30 min with 90% Methanol. Intracellular protein staining was performed with the following antibodies for 45 min at RT: phospho-p38 PE (sc-166182), p38 AF 647 (sc-81621), p-MK2a FITC (sc-293139) and MK2a AF 647 (sc-393609). All antibodies were purchased from Santa Cruz Biotechnology. The following isotype controls were used: lgG2a, k PE (Miltenyi Biotec), lgG1k AF 647 (BD) and lgG1k FITC (BD). Samples were measured on BD FACSCanto II.
  • Total histone protein extracts were isolated from CN and CN/AML iPSC- derived HSPCs of both CN/AML patients using the EpiQuikTM Total Histone Extraction Kit (Epigentek, catalog # OP-0006). Yield of the total histone protein extraction was meas ured by Quant-iTTM QubitTM protein assay kit (Thermo Fisher Scientific, catalog # Q33212). The inventors quantified trimethylated lysine 27 in histone H3 (H3K27me3) us ing the Histone H3 methylated Lys27 ELISA kit (Active Motif, catalog #53106) according to the standard protocol. The assay was performed in duplicates using 6 pg of total his tone protein extract per well.
  • Missense RUNX1 mutations are associated with trisomy 21 in CN/AML patients
  • the inventors generated iPSCs from two CN/AML patients harboring ELANE mutations, p.C151Y and p.G214R ( Figure 1B).
  • MNCs mononuclear cells isolated from peripheral blood (PB MNCs) of CN patient 1 at time of overt AML
  • the inven tors generated an iPSC clone carrying ELANE p.C151Y mutation only (CN1) and two iPSC clones carrying ELANE mutation and acquired nonsense CSF3R p.Q741X mutation in combination with missense RUNX1 p.R139G mutation and trisomy 21 (CN/AML1.1 and 1.2) ( Figures 3A and 3B).
  • Table 3 lists all sgRNA which are capable of knocking out the BAALC gene.
  • iPSC clones of CN patient 2 carrying ELANE p.G214R mutation only (CN2) or ELANE mutation and acquired CSF3R p.Q743X mutation (CN2 +CSF3R Q743X +/ ) were generated from BM MNCs isolated at AML stage ( Figure 1B; Figure 3A) Heterozygous frame-shift RUNX1 p.Glu175SerfsX7 mutation was introduced in CN2 +CSF3R Q743X +/ - iPSC clone using CRISPR/Cas9 (CN/AML2) ( Figure 1B; Figure 3E).
  • Array-CGH confirmed no chromosomal abnormalities in CN1 iPSC clone, trisomy 21 in CN/AML1.1 and 1.2 iPSC clones, and the additional gain of a part of chromosome 12 in CN/AML1.1 iPSC clone ( Figure 3B).
  • the chromosome 12 gain is a frequent finding during iPSC maintenance, reflecting high proliferation of these cells. No chromosomal abnormalities were detected in iPSC clones derived from CN patient 2 ( Figure 3B).
  • HSPCs were collected at day 14 of EB differenti ation and cultured on FLT3-L-secreting feeder cells.
  • the inventors observed a substantial increase in proliferation of CN/AML iPSC-derived HSPCs from both patients as compared to CN or healthy donor (HD) HSPCs ( Figures 6C and 6D).
  • Proliferation of HSPCs ex pressing mutated CSF3R was significantly higher than in CN cells, but much lower than in CN/AML cells ( Figures 6C and 6D).
  • Granulocytic differentiation was markedly reduced in CN- and CN -CSF3R iPSCs compared with HD iPSCs, and practically abolished in CN/AML iPSCs ( Figures 6E and 6F).
  • Morphological analysis of cytospin preparations re vealed almost no mature granulocytes in CN/AML samples ( Figures 6G and 6H).
  • CD34 + cells derived from CN iPSCs produced fewer numbers of CFU-Gs and CFU-GMs colonies as compared to HD iPSCs. These numbers were further decreased in CSF3R-mutated CN iPSCs and CN/AML iPSCs ( Figures 6I and 6J).
  • top genes that they found to be upregulated in primary CN/AML blasts BAALC (brain and acute leukemia, cytoplasmic), HPGDS (hematopoietic prostaglandin D synthase), CD34 and CD109 (own unpublished observations of transcriptome studies) might be also elevated in CN/AML iPSCs-derived HSPCs.
  • BAALC brain and acute leukemia, cytoplasmic
  • HPGDS hematopoietic prostaglandin D synthase
  • CD34 and CD109 (own unpublished observations of transcriptome studies) might be also elevated in CN/AML iPSCs-derived HSPCs.
  • BAALC, CD34, HPGDS and CD109 expression was also in creased in CD34 + cells derived from CN/AML1.1 and 1.2 iPSC lines compared with CN1 iPSCs, and BAALC mRNA was higher in CN/AML2 HSPCs as compared to CN2 cells ( Figures 7B and 7C).
  • BAALC protein was elevated in CN/AML HSPCs of both patients ( Figure 7D).
  • BAALC, but not CD109, RUNX1 or HPGDS knockout resulted in a dra matic induction of granulocytic differentiation and a significant reduction in proliferation of CN/AML1 iPSC-derived HSPCs ( Figures 8B-8E; Figure 9G).
  • the inventors ob served a striking increase in granulocytic differentiation along with a marked reduction in proliferation in CN/AML2 iPSC-derived HSPCs after BAALC KO ( Figures 8F-8I; Figure 9H).
  • BAALC KO did not affect granulocytic differentiation in HD iPSCs, but in prised granulopoiesis in CN iPSCs ( Figure 8J, Figures 9I-9K).
  • BAALC KO CRISPR/Cas9 KO effi ciency was around 80 %) led to a marked reduction of cell proliferation measured on day 14 of culture as compared to mock electroporated cells (Figure 8K).
  • the inventors compared the transcriptome of CN and CN/AML iPSCs- derived HSPCs from both patients.
  • Differential gene expression analyses using DESeq2 R package identified 132 up- and 570 down-regulated genes, as well as 570 up- and 1422 down-regulated genes between CN/AML and CN stages for patient 1 and patient 2, respectively (log2FC > 1 or ⁇ -1, adj. P-value ⁇ 0.05; Figures 10A and 10B; Figures 11A and 11B; Tables 4 and 5).
  • GSEA Gene set enrichment analysis
  • the inventors performed eXpression2Kinases analysis.
  • the inventors detected RUNX1 binding motifs, GATA1, GATA2 motifs, as well as AML-associated SUZ12 and EZH2 motifs (Figure 10E) to be significantly enriched in CN/AML cells.
  • TRIM28 and TP53 motifs were specifically enriched in CN/AML1, whereas CEBPB, NANOG, KLF4 in CN/AML2 ( Figure 10E).
  • KAA kinase enrichment analysis
  • the inventors predicted kinases that phosphorylate proteins regulated by the subnetwork of prioritized transcription factors.
  • enriched kinases including HIPK2, MAPK1/3/14, CSNK2A1, ERK1 and AKT1, were shared between both patients at the selected threshold (P-value ⁇ 10 8 ) (Fig ure 10F).
  • Unique enriched kinases e.g., CDK1, JNK1, ERK2 were detected in CN/AML2 only ( Figure 10F).
  • the inventors compared the transcriptomes of CN/AML iPSCs before and after BAALC KO.
  • the inventors identified 165 up- and 254 down-regulated genes between CN/AML1 and CN/AML1 BAALC KO, as well as 185 up- and 381 down-regulated genes between CN/AML2 and CN/AML2 BAALC KO (log2FC > 1 or ⁇ -1, adj. P-value ⁇ 0.05) ( Figures 12A and 12B; Figures 11D and 11 E; Tables 6 and 7).
  • BAALC KO led to a dramatic shift in the gene expression signature (Figure 12C,D).
  • GSEA revealed enrichment of oxidative phosphorylation, tricarboxylic acid cycle (TCA), p53 signaling and inhibition of platelet- specific genes in CN/AML1 sample as compared to BAALC KO ( Figure 12E).
  • T ranscription factor enrichment analysis of differentially ex pressed genes revealed RUNX1, GATA1/2, SUZ12 transcription factor binding motifs to be BAALC dependent in both patients.
  • AR, TCF3, RAD21, NANOG motifs were deregu lated in CN/AML1 only.
  • EGR1, STAT3 and ZC3H11A motifs were CN/AML2 specific (Fig ure 12G).
  • CSNK2A1, CDK1, GSK3B, HIPK2, MAPK14/p38a were among the top BAALC-dependent kinases in both patients ( Figure 12H).
  • CMAP Connectivity Map
  • the inventors assessed MK2a and p38a phosphorylation levels in CN and CN/AML iPSC-derived HSPCs as well as in CN/AML cells treated with CMPD1 and CN/AML BAALC KO cells.
  • CMPD1 treat ment or BAALC KO resulted in a reduction of MK2a but not p38a phosphorylation in CN/AML ( Figure 14A).
  • Basal levels of non-phosphorylated MK2a and p38a proteins re mained unchanged in all groups ( Figure 13B).
  • KLF-4 activator, APTO-253, in combination with 1 mM of AZD-6244 was either not effective in a dose of 10 nM of APTO-253, or was toxic at the 100 nM and 1000 nM concentrations (data not shown).
  • the inventors present here an in vitro model of stepwise leukemia devel opment in pre-leukemia bone marrow failure syndromes, exemplified by CN.
  • CRISPR/Cas9 gene-editing the inventors introduced gene alterations associated with pre-leukemia and leukemia conditions in iPSC lines of CN patients.
  • iPSCs recapitulating non-leukemic and leukemia conditions, the inventors were able to evaluate hematopoietic differentiation and intracellular signaling differences between these two conditions.
  • the inventors observed almost completely abrogated myeloid differentiation and elevated proliferation of CN/AML iPSCs compared with control or CN iPSCs.
  • the inventors generated iPSC lines carrying ELANE, CSF3R and, additionally, mutations and chromo somal abnormalities mimicking CN/AML phenotype: (1) missense RUNX1 mutation and trisomy 21 with two copies of mutated RUNX1, or (2) RUNX1 haploinsufficiency without trisomy 21. In both conditions, myeloid differentiation was severely abrogated, and prolif eration was elevated.
  • the inventors identified signaling pathways deregulated by both missense and frame-shift RUNX1 mutations in CN/AML cells or affected explicitly by each mutation.
  • SUZ12 and EZH2 are core PRC2 subunits, suggesting possible changes of H3K27me3 distribution in Rl//VX7-mutant cells.
  • the in ventors detected reduced H3K27me3 levels in CN/AML samples compared to CN sam ples. It would be interesting to further investigate a specific H3K27me3 distribution in CN/AML cells.
  • the inventors identified elevated BAALC levels in CN/AML blasts, and CRISPR/Cas9-mediated BAALC knockout inhibited proliferation, simultaneously inducing myeloid differentiation of CN/AML cells.
  • BAALC is upregulated in RL//VX7-mutated de novo AML and the inventors found that mutated RUNX1 induced BAALC expression in HD HSPCs.
  • High BAALC expression is associated with aggressive AML and poor progno sis.
  • the in ventors performed RNA sequencing of CD34 + cells derived from CN/AML and CN/AML BAALC KO cells.
  • BAALC KO led to a marked shift in gene expression in CN/AML cells in dependent of the RUNX1 mutation type.
  • the inventors detected oxidative phosphoryla tion, MYC-, T ⁇ Rb-, E2F- and G2M checkpoint- controlling pathways to be BAALC- dependent in CN/AML.
  • RUNX1, SUZ12, SOX2, GATA1/2, SMAD4, and SALL4 transcription factors as well as MAPK14, GSK3B, CSNK2A1, CDK1/2 kinases pathways were found to be downstream of BAALC in both CN/AML patients.
  • CMPD1 selective MK2a inhibitor
  • CMPD1 or alternative MK2a inhibitors, might be implemented in the treatment of CN/AML and BAALC h ' 9h de novo AML.
  • CN/AML and BAALC h ' 9h de novo AML patients have aggres sive leukemia with poor prognosis, and our findings represent a first step toward the es tablishment of advanced therapies for these leukemias.
  • CMPD1 treatment was partially effective in CN/AML BAALC KO cells.
  • BAALC expression was reduced upon CMPD1 treatment, suggesting a feedback regulation of BAALC expression by MK2a pathway.
  • iPSC-based in vitro model is reliable for investigating step wise leukemogenesis in pre-leukemia bone marrow failure syndromes.
  • Implementation of this model led to the characterization of high BAALC expression and elevated MK2a phos phorylation, as ultimate leukemia-causing events in CN/AML, and the identification of CMPD1 as a potential therapeutic drug for CN/AML and BAALC'' 9 ’ 9 de novo AML patients.
  • Inhibition of BAALC or treatment with MK2a inhibitors might prevent leukemia develop ment in CN/AML and eliminate RL//VX7-mutated BAALC hi9h de novo AML blasts ( Figure 141).
  • the findings of the inventors were confirmed in an in vivo model ( Figure 15).
  • the inventors provide a new compound and method for the prophylaxis and/or treatment of leukemia and/or the development of leukemia, in particular AML.

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Abstract

La présente invention concerne un composé destiné à être utilisé dans la prophylaxie et/ou le traitement de la leucémie et/ou le développement de la leucémie, une composition pharmaceutique pour la prophylaxie et/ou le traitement de la leucémie et/ou le développement de la leucémie, comprenant ledit composé, un procédé pour la prophylaxie et/ou le traitement de la leucémie et/ou le développement de la leucémie chez un être vivant, une molécule d'ARN guide (ARNg) unique pouvant être utilisée dans le procédé selon l'invention.
PCT/EP2022/060294 2021-04-20 2022-04-19 Composé et procédé pour la prophylaxie et le traitement de la leucémie WO2022223548A1 (fr)

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EP22716274.0A EP4326870A1 (fr) 2021-04-20 2022-04-19 Composé et procédé pour la prophylaxie et le traitement de la leucémie
US18/489,480 US20240122882A1 (en) 2021-04-20 2023-10-18 Compound and method for the prophylaxis and treatment of leukemia

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EP21169352.8 2021-04-20

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